Use of chenopodium formosanum (djulis) extract for antioxidation and improving skin appearance

ABSTRACT

A use of Chenopodium formosanum (Djulis) extract for resisting oxidation and improving skin condition. In particular, the Chenopodium formosanum (Djulis) extract is obtained with an aqueous solvent. The skin condition improved includes skin color, skin wrinkles, skin hydration, and skin texture.

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims the benefit of U.S. provisionalapplication Ser. No. 62/934,014, filed on Nov. 12, 2019, the entirecontents of which are hereby incorporated by reference.

REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(2138-US-UT001_SEQLIST_ST25.txt; Size: 2.3 KB; and Date of Creation:Nov. 12, 2020) is herein incorporated by reference in its entirety.

BACKGROUND Technical Field

The present invention relates to a use of Chenopodium formosanum(Djulis) extract, particularly to the use of Chenopodium formosanum(Djulis) extract for the use of antioxidation and improving skinappearance, including skin moisturizing, skin whitening, and skinwrinkle smoothing.

Related Art

Chenopodium formosanum (Djulis), also known as Taiwan quinoa, redquinoa, purple quinoa, and rainbow rice, is an annual herb and anendemic species found in Taiwan. It is a close relative of quinoa andbelongs to the Chenopodiaceae family and the genus Chenopodium.Distributed in the middle and low altitude mountain areas of central andsouthern Taiwan, it has more than a hundred years in aboriginal farminghistory. At present, the aboriginal tribe of Paiwan in Eastern Taiwanhas the most abundant seed resources. Red quinoa is edible, and itsseedlings, tender stems, and flower spikes can be added to cooking orboiling soup. Red quinoa seeds can also be ground into a powder to serveas raw materials for glutinous rice balls.

The protein content of red quinoa is twice that of rice. The fruit ofred quinoa is often dried, hulled, and mixed with other grains to serveas general edible rice. In food processing, different processing methods(steaming, microwave, roasting, frying, and puffing) can be used to makerice balls, biscuits, potato balls, puffs, mochi, muffins, and fragrantrice products. Red quinoa can serve as a garnish for plating and providerich nutritional value, and is gradually being loved by more and moreconsumers.

SUMMARY

The present invention discloses a red quinoa extract for improving skinconditions. The composition can be used as an antioxidant or for skinwhitening and skin moisturizing purposes.

In some embodiments, the red quinoa extract scavenges free radicals incells, wherein the red quinoa extract is extracted with an aqueoussolvent. In some embodiments, the cell is a skin cell.

In some embodiments, the red quinoa extract achieves the antioxidanteffect by enhancing the expression level of antioxidant-related genes.

In some embodiments, the antioxidant-related genes include at least oneof the SOD1 gene, the SOD2 gene, or the CAT gene.

In some embodiments, the red quinoa extract achieves the antioxidanteffect by increasing the abundance of antioxidant enzymes in a body.

In some embodiments, the Degrees Brix value of the red quinoa extract is7.0±5.

In some embodiments, the red quinoa extract is used to prepare acomposition for improving skin condition, wherein the red quinoa extractis extracted with an aqueous solvent.

In some embodiments, the red quinoa extract is used to prepare a skinwhitening composition, wherein the red quinoa extract is extracted withan aqueous solvent.

In some embodiments, the red quinoa extract achieves the skin whiteningeffect by inhibiting tyrosinase activity and/or reducing the amount ofmelanin production.

In some embodiments, the red quinoa extract is used to prepare a skinmoisturizing composition, and the red quinoa extract is extracted withan aqueous solvent.

In some embodiments, the red quinoa extract achieves the skin'smoisturizing effect by enhancing the expression of skinmoisturizing-related genes.

In some embodiments, skin moisturizing-related genes include at leastone of the TGM1 gene, the KRT1 gene, the KRT10 gene, and the KRT14 gene.

In some embodiments, the aqueous solvent is water or a solventcontaining organic acids. In some embodiments, the concentration of theorganic acid is 0.05%-1.00%.

In some embodiments, the red quinoa extract is formed by firstextracting red quinoa with an aqueous solvent and then undergoingglycolysis using a saccharification enzyme.

In summary, the red quinoa extract of any embodiment can be used toprepare an antioxidant composition. The red quinoa extract of anyembodiment can be used to prepare a composition that improves theexpression of antioxidant-related genes (such as the SOD1 gene, the SOD2gene, or the CAT gene) in cells. The red quinoa extract of anyembodiment can be used to prepare a composition for enhancingantioxidant enzymes in the body. The red quinoa extract of anyembodiment can be used to prepare a skin moisturizing composition. Thered quinoa extract of any embodiment can be used to prepare acomposition that improves the expression of moisturizing-related genes(such as the TGM1 gene, the KRT1 gene, the KRT10 gene, the KRT14 gene)in cells. The red quinoa extract of any embodiment can be used toprepare a skin whitening composition. The red quinoa extract of anyembodiment can be used to prepare a composition that inhibits tyrosineexpression. The red quinoa extract of any embodiment can be used toprepare a composition that inhibits melanin formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a method for preparing red quinoa extract.

FIG. 2 shows another embodiment of a method for preparing red quinoaextract.

FIG. 3 shows the tyrosine inhibition ratios of the experimental andcontrol groups in EXAMPLE 4.

FIG. 4 shows the melanin production ratios of the experimental andcontrol groups in EXAMPLE 5.

FIG. 5 shows the gene expression ratios of the antioxidant-related genesin the experimental and control groups in EXAMPLE 6.

FIG. 6 shows the gene expression ratios of the moisturizing-relatedgenes in the experimental and control groups in EXAMPLE 7.

FIG. 7 shows skin moisture retention ratios of the experimental andplacebo groups in EXAMPLE 8 after intake of health drinks with andwithout the red quinoa extract, respectively, for 4 weeks and 8 weeks.

FIG. 8 shows skin whiteness ratios of the experimental and placebogroups in EXAMPLE 8 after intake of health drinks with and without thered quinoa extract, respectively, for 4 weeks and 8 weeks.

FIG. 9 shows skin texture ratios of the experimental and placebo groupsin EXAMPLE 8 after intake of health drinks with and without the redquinoa extract, respectively, for 4 weeks and 8 weeks.

FIG. 10 shows crow's feet proportions of the experimental and placebogroups in EXAMPLE 8 after intake of health drinks with and without thered quinoa extract, respectively, for 4 weeks and 8 weeks.

FIG. 11 shows the amount of antioxidant enzyme present in the bodies ofthe experimental and placebo groups in EXAMPLE 8 after intake of healthdrinks with and without the red quinoa extract, respectively, for 4weeks and 8 weeks.

DETAILED DESCRIPTION

Some embodiments of the present invention are described below. Thepresent invention can be practiced in many different embodiments. Itshould be noted that the scope of the present invention is not limitedto the embodiments specified in the present specification.

Microsoft's Excel software is used for statistical analysis in thepresent invention. The data are expressed as mean±standard deviation(SD), and the differences between groups are analyzed by student'st-test. In the diagram, “*” represents a p-value smaller than 0.05, “**”represents a p-value smaller than 0.01, and “***” represents a p-valuesmaller than 0.001. The more “*”'s are present, the more significant isthe statistical difference.

The numerical values used in this article are approximate, and allexperimental data all include a margin of error of 10%. In someembodiments, the margin of error is 5%.

In this article, “wt %” refers to percentage by weight, and “vol %”refers to percentage by volume.

As used herein, the term “extract” refers to a product prepared throughextraction. The extract can be presented in the form of a liquidsolution or a form of a concentrate or essence containing no or littlesolvent.

As used herein, “raw red quinoa material” generally refers to theplant's seeds, wherein the seeds may include fresh red quinoa, dried redquinoa, or red quinoa processed by other physical methods. The physicalmethods may include chopping, dicing, or crushing. Any physical processthat renders the seed different from its original physical property isconsidered a physical method.

Referring to FIG. 1, in some embodiments, the red quinoa extract isextracted from raw red quinoa material with an aqueous solvent. In someembodiments, the red quinoa (Chenopodium formosanum (Djulis)) extract isobtained by sequentially performing the crushing procedure S01, heatingprocedure S02, filtering procedure S04, and concentrating procedure S06.

In some embodiments, the raw red quinoa material is hulled red quinoaseeds. In some embodiments, the red quinoa raw material may be fresh ordried red quinoa seeds.

In some embodiments, the crushing procedure S01 refers to grinding orcrushing the raw red quinoa material until it becomes powdered raw redquinoa material. A juicer, a blender, or a homogenizer can be used toperform the crushing procedure 01.

In some embodiments, the heating procedure S02 refers to mixing powderedraw red quinoa material with an aqueous solvent and heating it for afixed period. In some embodiments, heating refers to increasing the rawred quinoa material's temperature and its aqueous solvent to 50° C. to100° C. In some embodiments, a fixed period of time refers to 0.5 hoursto 3 hours. An example of an embodiment is mixing the powdered raw redquinoa material with an aqueous solvent and increasing the temperatureof the solution to 85° C. and maintaining such temperature for 1 hour.

In some embodiments, during the heating procedure S02, the weight ratioof the aqueous solvent to the raw red quinoa material ranges from is5:1-20:1. For example, the weight ratio of the aqueous solvent to theraw red quinoa material can be 10:1.

In some embodiments, the aqueous solvent is water or a water solventcontaining organic acids. In some embodiments, the concentration of theorganic acid is 0.05% to 1.00%. In some embodiments, the organic acidand its concentration are at an edible state. In some embodiments, theorganic acid may be citric acid, malic acid, tartaric acid, lactic acid,gluconic acid, acetic acid, but is not limited thereto.

An example of an embodiment is mixing 90 kg of raw red quinoa materialwith 900 kg of water and 0.63 kg of citric acid, heating them to 100° C.and maintaining them at 100° C. for 0.5 hours. Another example is mixing90 kg of raw red quinoa material with 900 kg of water and 0.7 kg ofcitric acid and then heating them to 85° C. and maintaining 85° C. for 1hour.

In some embodiments, the filtering procedure S04 refers to passing theheated (or cooled) raw red quinoa material and the aqueous solventthrough a filter to filter out the solids or large particles in thesolvent to form a filtrate. In some embodiments, the filter is a400-mesh filter.

Referring to FIG. 2, in some embodiments, between the heating procedure02 and the filtering procedure S04 is a cooling process S03. The coolingprocess S03 refers to cooling the processed raw red quinoa materials andits aqueous solvent to room temperature.

In some embodiments, the concentrating procedure S06 refers to obtaininga first filtrate by concentrating the filtrate obtained from thefiltering procedure S04 under reduced pressure (brand/model:BUCHI-Rotavapor R-100). In some embodiments, the first extract is thered quinoa extract. In some embodiments, the concentrating procedure S06provides an environment of 40° C.˜70° C. with reduced atmosphericpressure for concentrating. An example of an embodiment of the redquinoa extract is obtained by first crushing raw red quinoa materialinto powder through the crushing procedure S01, then undergoing theheating procedure S02, the filtering procedure, and the concentratingprocedures S06.

In some embodiments, between the filtering procedure S04 and theconcentrating procedure S06 is a glycolyzing procedure S05. During theglycolyzing procedure S05, a glycolysis enzyme is added to, stirredwith, and mixed with the filtrate obtained from the filtering procedureS04 and then allowed to stand for at least 1 hour to give a glycolyzedsolution. Glycolysis enzymes further enhance the effect of bioactiveingredients in the red quinoa extract. In some embodiments, theglycolysis enzymes may be α-amylase, [beta]-amylase, glucoamylase,isoamylase, or a mixture of at least one. In some embodiments, theglycolysis enzymes may be glucose amylase (glucan1,4-alpha-glucosidase). In some embodiments, the filtrate obtained fromthe filtering procedure S04 is added with a 0.06% amount of starchsaccharification enzymes. In some embodiments, the S05 glycolyzingprocedure includes adding a 0.06% amount of a starch saccharificationenzyme to the filtrate obtained from the filtering procedure 04 and thenheated to and maintained at 55° C. maintained for more than 1 hour toobtain a glycolyzed solution.

In other embodiments, the S06 concentrating procedure concentrates theglycolyzed solution obtained from the glycolyzed solution S05 withreduced atmospheric pressure.

In some embodiments, after the concentrating procedure S06 is a secondfiltering procedure S07. The second filtering procedure S07 refers topassing the glycolyzed solution obtained by the glycolyzing procedureS06 through a 400-mesh filter to obtain the red quinoa extract. Anexample of an embodiment of red quinoa extract is obtained bysequentially going through the crushing procedure S01, the heatingprocedure S02, the filtering procedure S04, the glycolyzing procedureS05, the concentrating procedure S07, and the second filtering procedureS07.

In some embodiments, the red quinoa extract is used to prepare anantioxidant composition, wherein the red quinoa extract achieves theantioxidating effect by promoting the expression of genes related toantioxidation, and wherein the red quinoa extract is extracted with anaqueous solvent. In some embodiments, the red quinoa extract achievesthe antioxidating effect by increasing antioxidant enzymes in a body.

In some embodiments, antioxidation refers to the slowing down of or theprevention of oxidation. Oxidation refers to a chemical reaction inwhich one or more elections are transferred from one matter to another.This chemical reaction may generate a radical species and consequentlystart a chain reaction. When a chain reaction occurs in a cell, the cellwill be susceptible to damage or apoptosis. In some embodiments, the redquinoa extract removes free radicals, terminate the chain reaction, orinhibits other oxidation reactions. In some embodiments, the radicalspecies in a cell are generated due to light, chemicals, or the naturalprocess of aging.

In some embodiments, the antioxidant-related genes include at least oneof the SOD1 gene (GeneID: 6647), the SOD2 gene (GeneID: 6648), or theCAT gene (GeneID: 847).

As described above, superoxide anions, hydrogen peroxides, and otherfree radicals can damage skin cells. Proteins transcribed by the SOD1gene and the SOD2 gene (superoxide dismutase) break down superoxide intohydrogen peroxide. The protein transcribed by the CAT gene breaks downhydrogen peroxide into water and oxygen. The genes transcribe proteinsthat are important for the removal of free radicals. Thus, the moreincreased is the expression of these genes in a cell, the better thecell's antioxidant capacity is.

In other words, by promoting the expression of the aforementionedantioxidation-related genes, the red quinoa extract effectively enhancescellular antioxidation capacity, improves cellular resistance to freeradicals, thus protecting skin cells and improving skin condition.

In some embodiments, a concentration of 2 mg/mL of red quinoa extractpromotes expression levels of antioxidation-related genes, removes freeradicals, and increases cellular antioxidation capacity.

In some embodiments, a red quinoa extract having a Degrees Brix of 7.0or more improves the expression levels of antioxidation-related genes,removes free radicals, and increases cellular antioxidation capacity.

In some embodiments, a red quinoa extract is used to prepare a skinwhitening or brightening composition, wherein the red quinoa extract isachieved the skin whitening or brightening by inhibiting melaninproduction or inhibiting tyrosine production, wherein the red quinoaextract obtained from an aqueous solvent.

The production of melanin darkens the skin, and during the process ofmelanin production, tyrosinase plays a crucial role. Tyrosinase is arate determine enzyme that regulates melanogenesis. Specifically,tyrosinase is involved in two reactions during melanin synthesis: (1)the conversion of a phenolic hydroxyl group into a single diphenol, (2)the oxidation of diphenols to quinones. The enzyme tyrosinase is presentin skin melanocytes and is encoded in the TYR gene.

In some embodiments, the red quinoa extract suppresses tyrosinaseformation to inhibit, prevent, or slow down melanin production in theskin, which in turn whitens or brightens the skin.

In some embodiments, a concentration of 1 mg/mL of red quinoa extract isused to whiten and brighten skin.

In some embodiments, red quinoa extract with a Degrees Brix of 7.0 ormore is used to whiten and brighten skin.

In some embodiments, the red quinoa extract is used to prepare a skinmoisturizing composition, wherein the red quinoa extract is extractedwith an aqueous solvent, and wherein the red quinoa extract s extractedquinoa through ascension the amount of moisture associated geneexpression to achieve the effect of moisturizing the skin.

During daily activities, the skin is prone to water loss due to theweather, the external environment, or even aging. This water loss isaccelerated if the gaps between skin cells are increased. Thisacceleration of water loss results in the reduction of skin brightnessand moisture retention capacity.

In some embodiments, moisture-related genes include the gene TGM1(GeneID: 7051), the KRT1 gene (GeneID: 3848), the KRT10 gene (GeneID:3858) and the gene KRT14 (GeneID: 3861).

The KRT1 gene (GeneID: 3848), the KRT10 gene (GeneID: 3858), and theKRT14 gene (GeneID: 3861) encodes the protein keratin, which helpsmaintain the skin's protective ability.

Specifically, the KRT1 gene, the KRT10 gene, and the KRT14 gene encodethe proteins keratin 1, keratin 10, and keratin 14. Keratin is a fibrousprotein that forms the main structure of keratinocytes. Keratin 1,Keratin 10, and Keratin 14 link together to form intermediate filamentsthat act as strong fiber networks and provide strength and elasticity tothe skin. They protect the skin from friction and other physical damagesand pack skin cells more closely together, resulting in the reduction ofwater loss via gaps between skin cells and, consequently, the skin'smoisturization.

In other words, red quinoa extract promotes the expression levels of theaforementioned genes and thereby improves skin conditions.

In some embodiments, a concentration of 2 mg/mL or more of red quinoaextract enhances the skin's ability to retain moisture.

In some embodiments, a Degrees Brix of 7.0 or more of red quinoa extractenhances the skin's ability to retain moisture.

In some embodiments, the red quinoa extract is used to prepare a wrinklesoothing and/or skin texture improving composition.

In some embodiments, a concentration of more than 10 wt % of red quinoaextract smoothes out wrinkles and/or improves skin texture.

In some embodiments, a Degrees Brix of 7.0 or more of red quinoa extractenhances the skin's ability to retain moisture.

In some embodiments, the composition is a pharmaceutical composition. Inother words, this pharmaceutical composition comprises an effectiveamount of red quinoa extract.

In some embodiments, the pharmaceutical composition can be manufacturedinto a medicament or formula suitable for non-parenteral or oraladministration by using ordinary skills in the art. These medicaments orformulas may include, but are not limited to: tablets, troches,lozenges, pills, capsules, dispersible powders, granules, solutions,suspensions, emulsions, syrups, elixirs, slurries, or other medicamentsor formulas having similar functions.

In some embodiments, the pharmaceutical composition can be manufacturedinto a medicament or formula suitable for non-parenteral or topicaladministration by using ordinary skills in the art. These medicaments orformulas include but are not limited to: injections, sterile powders,external preparations (external preparation), or other medicaments orformulas having similar functions. In some embodiments, thepharmaceutical composition may be administered through one of thefollowing non-parenteral routes: subcutaneous injection, intraepidermalinjection, intradermal injection, or intralesional injection.

In some embodiments, the pharmaceutical composition may further containa widely used pharmaceutically acceptable carrier. In some embodiments,the pharmaceutically acceptable carrier contains one or more of thefollowing reagents: solvents, buffers, emulsifiers, suspending agents,decomposers, disintegrating agents, dispersing agents, binding agents,excipients, stabilizers, chelating agents, diluents, gelling agents,preservatives, wetting agents, lubricants, absorption delaying agents,liposomes or other pharmaceutically acceptable carriers of similarfunctions. The selection and quantification of these agents are routinepractice and techniques for people having ordinary skill in the art.

In some embodiments, the pharmaceutically acceptable carrier comprisesone or more of the following solvents: water, normal saline, phosphatebuffered saline (PBS), an aqueous solution containing alcohol.

In some embodiments, the composition may be a food composition. In someembodiments, the food composition may be a food product or a foodadditive. The food additive refers to a matter that is added tomanufacture a food product with conventional methods or is added duringthe production process of a food product. This food product can beformulated with edible materials, and the food product can be consumedby humans or animals.

In some embodiments, the food product may be but are not limited to:drinks (beverages), fermented foods, bakery products, health foods, anddietary supplements.

EXAMPLE 1 Preparation of the Red Quinoa ((Chenopodium formosanum(Djulis))) Extract

Dried red quinoa fruits (husk included) are crushed during the crushingprocedure with an Osterizer 10 speed blender. Next, the crushed driedquinoa fruits undergo the heating procedure in which water serves as theaqueous solvent. The weight ratio of the crushed dried quinoa fruits tothe aqueous solvent is 1:10 and the heating procedure is maintained at85±5° C. for 60 minutes during which the extraction takes place. Then,the solution obtained from the heating procedure is cooled to roomtemperature (25° C.) and is passed through a 400-mesh filter to form afiltrate. Finally, the filtrate is concentrated under sub-atmosphericpressure at 60° C. until the filtrate reaches a Degrees Brix value of7.0±5 to give the disclosed red quinoa extract.

In some embodiments, a glycolyzing procedure is added between thefiltering procedure and the concentrating procedure. That is, thefiltrate is added with a 0.06% starch saccharification enzyme andundergoes a glycolyzing process at 55° C. for 1 hour to obtain aglycolyzed solution. Hereto, a commercially available AMG 300L enzyme isused as the starch saccharification enzyme.

In some embodiments, the aqueous solvent further includes an organicacid, wherein the organic acid is citric acid. The amount of citric acidis 0.05 wt % of the overall solvent.

EXAMPLE 2 Quantifying Flavonoid Content

Using water, dilute the red quinoa extract obtained from EXAMPLE 1ten-fold in terms of volume and take 200 μL of the diluted solution to atest tube. Add 5 wt % of sodium nitrite (Sigma, product No. 31443) tothe test tube and allow the reaction to stand for 6 minutes.

Next, add 10 wt % of aluminum nitrate (Alfa Aesar, product No. 12360) tothe test tube solution, and let the reaction stand for 6 minutes. Then,after adding 2 mL of 4 wt % aqueous sodium hydroxide (Macron, productNo. 7708-10) to the solution, add 1.4 mL of water to the test tube andmix evenly to obtain the reaction solution. Take 200 μL of reactionsolution into a 96-well reaction plate and detect the absorbance valueat a wavelength of 500 nm using a spectrophotometer (Jasco, ModelV-730).

Then, prepare standard solutions for the calibration curve: Hereto,rutin (ChromaDex, Product No. ASB-00018440) equivalents are used toexpress the relative content of total flavonoids. First, a concentrationof 200 μg/mL of rutin methanol solution is used as the standardsolution. Amounts of 0 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μLof the standard solution were added to different tubes. Water is thenadded to the test tubes, such that the total volume of each tube is 1200μL. Next, for the solution in each tube, 200 μL is taken to mix with 200μL of 5 wt % aqueous sodium nitrite solution in a new test tube wherethe reaction is allowed to stand or 6 minutes. Followed by the additionof 200 μL 10 wt % aqueous aluminum nitrate solution, the reaction isallowed to stand for 6 minutes. Then add 2 mL of 4 wt % sodium hydroxideaqueous solution for homogenization. Next 1.4 mL of water is added tothe tube and mixed to obtain a reaction solution. Finally, take 200 μLof the reaction solution into a 96-well reaction plate and detect theabsorbance value at a wavelength of 500 nm using a spectrophotometer(Jasco, Model V-730). Plot the absorbance values of the six differentconcentrations of rutin to give a calibration curve.

The calibration curve of absorbance can be used to convert absorbancevalue into total flavonoid content. Thereto, an estimation of totalflavonoids of the red quinoa extract obtained from EXAMPLE 1 is 1000ppm.

Flavonoids are not known to have the uses of improving skin condition orhaving antioxidative or skin whitening properties. But since it is knownthat flavonoids may have other uses such as the prevention ofcardiovascular disease, the high total flavonoid content in the presentdisclosure indicates that the red quinoa extract can also have a varietyof complex applications. Further, in some embodiments, the totalflavonoid content can be used as an endpoint criterion for theconcentrating procedure.

EXAMPLE 3 Quantifying Total Polyphenol Content

Using water, dilute the red quinoa extract obtained from EXAMPLE 1ten-fold in terms of volume and take 100 μL of the diluted solution to atest tube. Then, add 500 μL of Folin-Ciocalteu's phenol reagent (Merck,Product No. 1.09001.0100), mix well, and leave to stand for 3 minutes.Then add 400 μL of 7.5 wt % sodium carbonate (Sigma, Product No. 31432)to the test tube, mix well, and allow it to react for 30 minutes. Afteroscillation (Vortex) and ensuring that no air bubbles are present, take200 μL of the solution and record its absorbance value at a wavelengthof 750 nm.

Next, prepare the standard solutions to produce the calibration curve.Hereto, gallic acid equivalents are used to express the relative contentof total polyphenols. Thereto, concentrations of 0 μL/mL, 20 μL/mL, 40μL/mL, 60 μL/mL, 80 μL/mL, and 100 μL/mL of gallic acid (Sigma, productNo. G7384) standard solution were made, where 100 μL of each solutionwere taken to a new test tube, respectively. For each test tube, 500 μLof Folin-Ciocalteu's phenol reagent is added, mixed well, and left tostand for 3 minutes. Followed by the addition of 400 μL of 7.5 wt %aqueous sodium carbonate solution, the test tube solution is mixed welland allowed to react for 30 minutes. After oscillation (Vortex) andensuring that no air bubbles are present, take 200 μL of the solutionand record its absorbance value at a wavelength of 750 nm. Plot theabsorbance values of the six different concentrations of gallic acidsolution to give a calibration curve.

The calibration curve of absorbance can be used to convert absorbancevalue into total polyphenol content. Thereto, an estimation of totalpolyphenols of the red quinoa extract obtained from EXAMPLE 1 is 400ppm.

Polyphenols are not known to have the uses of improving skin condition,or having antioxidative or skin whitening properties. But since it isknown that polyphenols may have other uses such as the prevention ofcardiovascular disease, the high total polyphenol content in the presentdisclosure indicates that the red quinoa extract can also have a varietyof complex applications. Further, in some embodiments, the totalpolyphenol content can be used as an endpoint criterion for theconcentrating procedure.

EXAMPLE 4 Inhibition of Tyrosine Production

Tyrosinase has a key role in the synthesis of melanin. Tyrosinasecatalyzes the conversion of tyrosine to L-Dopa, which is furtherproduced into Dopaquinone and then to melanin. Thus, by detecting theactivity of tyrosinase, melanocytes' ability to produce melanin can beinferred. When the activity of tyrosinase is low, the melanin productioncapacity of melanocytes is relatively reduced.

Materials and Equipment

-   -   1. Cell line: B16F10 mouse melanoma cells, purchased from        American Type Culture Collection (American Type Culture        Collection, ATCC®, No. 6475), hereinafter referred to as B16F10        cells.    -   2. Cell culture medium: Basal medium containing 10 vol % FBS        (fetal bovine serum, available from Gibco), wherein the basal        medium is Eagle's minimum essential medium (MEM) (Gibco, Catalog        No. 15188-319) with the addition of 1 mM sodium pyruvate        (Gibco), 1.5 g/L sodium bicarbonate (Sigma), and 0.1 mM        non-essential amino acid solution (Gibco).    -   3. Phosphate Buffered Saline solution (PBS solution): purchased        from Gibco, Catalog No.: 10437-028.    -   4. Kojic acid, purchased from Sigma-Aldrich, product No.        K3125-5G.    -   5. Radioimmunoprecipitation protein lysis buffer (RIPA Lysis        Buffer (PMSF)): purchased from Sigma-Aldrich, product No.        10837091001.    -   6. Bio-rad Protein Assay, available from Biotek, Item Epoch.    -   7. 10 mM L-dopa: obtained by dissolving 0.8 mg of L-dopa        (Sigma-Aldrich, product No. D9628-5G) into 5 mL of 0.1 mM, pH        7.0 PBS.    -   8. Trypsin: 10× Trypsin-EDTA (Gibco) diluted 10-fold in 1×PBS        solution.    -   9. Red quinoa extract: prepared by the method detailed in        EXAMPLE 1.

Experimental Procedure:

Seed 1.5×10⁵ B16F10 cells with 2 mL of medium in each well of a 6-wellplate and incubate at 37° C. for 24 hours.

Then, carefully remove the culture medium without interfering with theattachment of cells.

This experiment is divided into three groups: the control group,experimental group A, and experimental group B. The control group isadded with 2 mL of fresh medium and incubated for 48 hours; experimentalgroup A is added with 2 mL of 1 mg/mL red quinoa extract and incubatedfor 48 hours; experimental group B is added with 2 mL of 0.5 mg/mL redquinoa extract, and incubated for 48 hours. The red quinoa extractsamples in experimental groups A and B were obtained by mixing 2 mL ofmedium with 14.3 μL and 28.6 μL of the red quinoa extract prepared fromEXAMPLE 1.

Subsequently, the medium is removed and washed with 1×PBS (Gibco) twice.Trypsin-EDTA was added to the wells and to react for 3 minutes. Thecells were then retrieved from the suspension and taken into 15 mLcentrifuge tubes, where they were centrifuged for 5 minutes at 400×g topellet the cells.

Then, the precipitated cells were washed twice with 1×PBS, suspendedwith 200 μL cell lysate, vortexed, and centrifuged for 20 min at12,000×g.

Transfer the supernatant to 1.5 mL centrifuge tubes, where the proteinconcentrations are detected:

Bio-rad dye is mixed with deionized water (volume ratio 1:4) anddistributed into 7 microcentrifuge tubes, each having 500 μL of thesolution. Then, the microcentrifuge tubes were added with 10 μL, 8 μL, 6μL, 4 μL, 2 μL, 1 μL, and 0 μL of 2 mg/mL BSA, respectively to preparethe standard protein concentration sample. Next, 2 μL of supernatant isadded to each microcentrifuge tube for testing. The microcentrifugetubes were then added to the wells of a 96 well plate where each well isadded with 200 μL of protein assay reagent. Then, the absorbance valuewas measured at 595 nm using the ELISA reader (BioTek).

Next, 400 μg of protein is added to each well Then, 90 μL cell lysate isadded to each well, including the control group. Under a darkenvironment, add 10 μL, 10 M of L-Dopa at 37° C. and observe thesuspension once every 10 minutes, until the suspension turns black.Then, measure the absorbance value at 405 nm.

Tyrosinase inhibition is calculated with the following formula:

Tyrosinase Inhibition (%)=(OD sample/OD control)×100%.

The results are then analyzed with Microsoft EXCEL software using itsStudent t-test statistical analysis.

Referring to FIG. 3, the tyrosinase inhibition is improved in the groupcontaining the red quinoa extract and increases with the red quinoaextract concentration. Specifically, experimental group B (0.5 mg/mL ofred quinoa extract) has a 30.4% higher tyrosine inhibition rate thanexperimental group A (1 mg/mL of red quinoa extract), which has a 42.6%higher tyrosine inhibition rate than the control group. Thus, the redquinoa extract disclosed herein reduces tyrosinase activity, which, inturn, reduces the formation of melanin. This shows that the red quinoaextract disclosed here can be used to reduce skin spots, improve skincondition, whiten the skin.

EXAMPLE 5 Reduction of Melanin Content

This example demonstrates the melanin content reducing capability of thered quinoa extract disclosed herein by quantifying the difference inmelanin content after treating B16F10 melanoma cells with the red quinoaextract disclosed herein with ELISA reader (enzyme-linked immunosorbentassay reader).

Materials and Equipment

-   -   1. Cell line: B16F10 mouse melanoma cells, purchased from        American Type Culture Collection (American Type Culture        Collection, ATCC®, No. 6475), hereinafter referred to as B16F10        cells.    -   2. Cell culture medium: Basal medium containing 10 vol % PBS        (fetal bovine serum, available from Gibco), wherein the basal        medium is Eagle's minimum essential medium (MEM) (Gibco, Catalog        No. 15188-319) with the addition of 1 mM sodium pyruvate        (Gibco), 1.5 g/L sodium bicarbonate (Sigma), and 0.1 mM        non-essential amino acid solution (Gibco).    -   3. Phosphate Buffered Saline solution (PBS solution): purchased        from Gibco, Catalog No.: 10437-028.    -   4. 1N NaOH (Sigma, Product No. 221465).    -   5. ELISA reader (BioTek, FLx 800).    -   7. Blue Light (wavelength: 400 nm-500 nm).    -   6. Red quinoa extract: prepared from EXAMPLE 1.

Experimental Procedure

This experiment is divided into two groups with three replicates: theexperimental group and the control group:

-   -   1. Seed 1.5×10⁵ B16F10 cells with 2 mL of medium in each well of        a 6-well plate.    -   2. Incubate plate at 37° C. for 24 hours.    -   3. Carefully remove culture medium without interfering with        attachment of cells.    -   4. The experimental group is added with 2 mL of 0.5 mg/mL red        quinoa extract and incubated for 48 hours. The red quinoa        extract sample was obtained by mixing 2 mL of medium with 14.3        μL of the red quinoa extract prepared from EXAMPLE 1.

The control group is added with 2 mL of fresh medium and incubated for48 hours.

-   -   5. The experimental group is exposed to blue light at room        temperature (25±5° C.) for 3 hours.

The control group is moved to a dark room and allowed to stand at roomtemperature for 3 hours.

-   -   6. Next, incubate cells at 37° C. for 48 hours.    -   7. The culture medium is removed and the cells were washed with        PBS solution two times.    -   8. 200 μl Trypsin-EDTA (10×) (Gibco; Catalog No. 15400-054) is        added to each well for 3 minutes. Then, 6 mL of culture medium        is added to terminate the reaction. Collect the suspended cells        to a 15 ml centrifuge tube and centrifuge at 400×g for 5 minutes        to pellet the cells.    -   9. After washing with PBS twice, resuspended the cells with 200        μL PBS.    -   10. The cell suspension is frozen in liquid nitrogen for 10        minutes and then left at room temperature for about 30 minutes        until completely thawed.    -   11. After thawing is complete, the tubes were centrifuged at        12,000 g for 3 minutes.    -   12. After removing the supernatant, use 120 μL of 1N sodium        hydroxide solution to precipitate the cells and put the test        tube in a hot plate at 60° C. for 1 hour to obtain samples for        detection.    -   13. Take 100 μL of the sample into a 96-well plate, and measure        optical density at 450 nm with the ELISA reader.

Experimental Results

Melanin content of the groups are calculated with the following formula:

Melanin Content (%)=(OD450 Experimental/OD450 Control)×100%.

Since the experiment was carried out in triplets, the values wereaveraged, as shown in FIG. 4.

As shown in FIG. 4, the melanin content of the experiment group is 29.7%lower than the control group, indicating that the red quinoa extractdisclosed herein has can effectively reduce the production of melanin inmelanocytes.

EXAMPLE 6 Enhanced Expression of Antioxidation-Related Genes

This example uses an RNA extraction kit, reverse transcriptase, a KAPASYBR® FAST qPCR reagent kit, and quantitative PCR to determine theexpression of antioxidation-related genes in human skin fibroblasts(CCD-966sk) after being treated by the red quinoa extract disclosedherein.

Materials and Equipment

-   -   1. Cell line: Human skin fibroblasts CCD-966sk (BCRC No. 60153).    -   2. Cell culture medium: Basal medium containing 10 vol % FBS        (fetal bovine serum, available from Gibco), wherein the basal        medium is Eagle's minimum essential medium (MEM) (Gibco, Catalog        No. 15188-319) with the addition of 1 mM sodium pyruvate        (Gibco), 1.5 g/L sodium bicarbonate (Sigma), and 0.1 mM        non-essential amino acid solution (Gibco).    -   3. RNA extraction reagent kit (Geneaid, Taiwan, Lot No.        FC24015-G).    -   4. Reverse Transcriptase (SuperScript® III Reverse        Transcriptase) (Invitrogen Corporation, USA, No. 18080-051).    -   5. Target gene primers: the SOD2 gene and the GAPDH gene        (internal control group).    -   6. KAPA SYBR® FAST qPCR reagent set (purchased from Sigma, USA,        No. 38220000000)    -   7. AB1 StepOnePlus™ Real-Time PCR system (Thermo Fisher        Scientific Inc., USA).    -   8. Red quinoa extract: prepared from EXAMPLE 1.

Experimental Procedure

The cell culture experiment process is as follows:

This experiment is divided into two groups with three replicates: theexperimental group and the control group:

-   -   1. Seed 1.5×10⁵ CCD-966sk cells with 2 mL of medium in each well        of a 6-well plate and incubate at 37° C. for 16 hours.    -   2. Experimental group: per ml culture broth containing a        red-quinoa extract prepared 28.6 μL of a sample obtained by the        embodiment (i.e., at a concentration of 1 mg/mL) is prepared to        contain extract broth, the CCD-966sk cell extracts containing        replaced broth culture is continued.    -   3. The experimental group is added with 2 mL of 1 mg/mL red        quinoa extract and incubated for 6 hours. The red quinoa extract        sample was obtained by mixing 2 mL of medium with 28.6 μL of the        red quinoa extract prepared from EXAMPLE 1.

The control group is added with 2 mL of fresh medium and incubated for 6hours

-   -   4. After incubation, lysate the cells of both the experimental        and control groups using the reagents provided in the RNA        extraction kit.

The polymerase chain reaction experiment process is as follows:

-   -   a. Collect the RNA of the cells in both groups using the RNA        extraction kit.    -   b. Next, take 2000 nanograms (ng) of the RNA extracted in each        group as a template, and use the primers listed in Table 1 to        reverse-transcribe cDNA using SuperScript® III reverse        transcriptase.    -   c. Use the primers in Table 1 to conduct quantitative real-time        reverse transcription polymerase chain reaction with        StepOnePlus™ Real-Time PCR system and KAPA SYBR FAST. This        allows the observation of the gene expression levels of the        experimental group and the control group. The parameters for the        PCR are 95° C./1 s, 60° C/20 s, 40 loops    -   d. The relative gene expression levels are determined by the        2^(−ΔΔCt) method. The “relative gene expression levels” are        defined as the fold change of the experimental groups RNA        expression level to that of the control group. The GADPH gene is        served as the internal control of the reference gene cycle        threshold (Ct). Fold change is calculated according to the        following formula:

ΔCt=Ct_(experimental group target gene/control group target gene)−Ct_(TBP)

ΔΔCt=ΔCt of experimental group−ΔCt gene of the control group

Fold change=2^(−ΔΔCt average)

-   -   5. Finally, the standard deviation and a one-tailed Student        t-test analysis are conducted with Microsoft Excel to determine        whether a significant difference exists (* p value<0.05        statistically; ** p value<0.01; *** p value<0.001). The SOD2        gene's corresponding primer is SOD2-F and SOD2-R while the gene        corresponding to the GADPH is GADPH-F primers and GADPH-R.

TABLE 1 Primer Sequence No. Sequence SOD2-F SEQ ID NO: 1AGAGTGGACCAACTGAAGAGT SOD2-R SEQ ID NO: 2 ATTCTCTGCATTTGTCCGCTT GADPH-FSEQ ID NO: 3 TCCTACTTGGACAAAGTTCGGG GADPH-R SEQ ID NO: 4CCCCTGATGTGAGTTGCCA

Experimental Results

FIG. 5. shows the relative expression levels of theantioxidation-related genes of the experiment and control group.

From FIG. 5., the relative expression level of the SOD2 gene in theexperimental group is 2725%. In other words, compared with the controlgroup, the expression level of the antioxidation-related SOD2 gene inthe experimental group increased 2625%, reaching statisticalsignificance. Thus, the red quinoa extract disclosed herein has freeradical scavenging properties and can function as an antioxidant.

EXAMPLE 7 Enhanced Expression of Moisture-Related Genes

This example uses an RNA extraction kit, reverse transcriptase, a KAPASYBR® FAST qPCR reagent kit, and quantitative PCR to determine theexpression of moisture-related genes in human epidermal keratinocytes(HPEK-50) after treatment of the red quinoa extract disclosed herein.

Materials and Equipment

-   -   1. Keratinocyte serum-free medium (Keratinocyte-SFM; available        from Thermo, part number 17005042).    -   2. Human epidermal keratinocytes (hereinafter as HPEK-50 cells        or keratinocytes; available from CELLnTEC).    -   3. RNA extraction reagent kit (Geneaid, Taiwan, Lot No.        F024015-G).    -   4. Reverse Transcriptase (SuperScript® III Reverse        Transcriptase) (Invitrogen Corporation, USA, No. 18080-051).    -   5. Target gene primers: the TGM1 gene, the KRT1 gene, the KRT10        gene, and the TBP gene (internal control group).    -   6. KAPA SYBR® FAST qPCR reagent set (purchased from Sigma, USA,        No. 38220000000)    -   7. ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher        Scientific Inc., USA).    -   8. Red quinoa extract, prepared from EXAMPLE 1.

Experimental Procedure

The cell culture experiment process is as follows:

This experiment is divided into two groups with three replicates: theexperimental group and the control group:

-   -   1. Seed 1.5×10⁵ HPEK-50 cells with 2 mL of medium in each well        of a 6-well plate and incubate at 37° C. for 16 hours.    -   2. The experimental group is added with 2 mL of 2 mg/mL red        quinoa extract, and incubated for 6 hours. The red quinoa        extract sample was obtained by mixing 2 mL of medium with 57.2        μL μL of the red quinoa extract prepared from EXAMPLE 1.

The control group is added with 2 mL of fresh medium and incubated for 6hours

-   -   3. After incubation, lysate the cells of both the experimental        and control groups using the reagents provided in the RNA        extraction kit.    -   4. The polymerase chain reaction experiment process is as        follows:    -   a. Collect the RNA of the cells in both groups using the RN A        extraction kit.    -   b. Next, take 2000 nanograms (ng) of the RNA extracted in each        group a template, and use the printers listed in Table 1 to        reverse transcribe cDNA using SuperScript® III reverse        transcriptase.    -   c. Use the primers in Table 2 to conduct quantitative real-time        reverse transcription polymerase chain reaction with        StepOnePlus™ Real-Time PCR system and KAPA SYBR FAST. This        allows the observation of the gene expression levels of the        experimental group and the control group. The parameters for the        PCR are: 95° C./1 s, 60° C./20 s, 40 loops    -   d. The relative gene expression levels are determined by the        2^(−ΔΔCt) method. The “relative gene expression levels” are        defined as the fold change of the experimental group's RNA        expression level to that of the control group. The TBP gene is        served as the internal control of the reference gene cycle        threshold (Ct). Fold change is calculated according to the        following formula:

ΔCt=Ct_(experimental group target gene/control group target gene)−Ct_(TBP)

ΔΔCt=ΔCt of experimental group−ΔCt gene of the control group

Fold change=2^(−ΔΔCt average)

5. Finally, the standard deviation and a one-tailed Student t-testanalysis is conducted with Microsoft Excel to determine whether asignificant difference exists (* p value<0.05 statistically; ** pvalue<0.01; *** p value<0.001). The TGM1 gene's corresponding primerpair is TGM1-F and TGM1-R, the KRT1 gene's corresponding primer pair isKRT1-F and KRT1-R, the KRT10 gene's corresponding primer pair is KRT10-Fand KRT10-R, the TBP gene's corresponding primer pair is TBP-F andTBP-R.

TABLE 2 Primer Sequence No. Sequence TGM1-F SEQ ID NO: 5GATCGCATCACCCTTGAGTTAC TGM1-R SEQ ID NO: 6 GCAGGTTCAGATTCTGCCC KRT1-FSEQ ID NO: 7 AGAGTGGACCAACTGAAGAGT KRT1-R SEQ ID NO: 8ATTCTCTGCATTTGTCCGCTT KRT10-F SEQ ID NO: 9 TATACTTGGACAAAGTTCGGG KRT10-RSEQ ID NO: 10 CCCCTGATGTGAGTTGCCA TBP-F SEQ ID NO: 11TATAATCCCAAGCGCTTTTGC TBP-R SEQ ID NO: 12 GCTGGAAAACCCAACTTCTG

Experimental Results

FIG. 6. shows the relative expression levels of the moisture-relatedgenes of the experiment and control group.

From FIG. 5, the relative expression levels of the TGM1 gene, the KRT1gene, and the KRT10 gene are 234%, 432% and 435%, respectively. In otherwords, compared with the control group, the expression level of the TGM1gene, the KRT1 gene, and the KRT10 gene in the experimental groupincreased 134%, 332%, and 335%, respectively, all reaching statisticalsignificance. Thus, the red quinoa extract disclosed herein can preventskin water loss, moisturize the skin and improve skin function.

EXAMPLE 8 Clinical Trials—Antioxidation and Skin Condition Improvement

This example demonstrates the antioxidation and skin condition,improving properties of the red quinoa extract disclosed herein byadministering drinks containing the red quinoa extract disclosed in thepresent invention to human subjects.

Drinking Sample: Beverage containing the presently disclosed red quinoaextract 50 g/bottle, which comprises: red quinoa extracted 10 wt %,apple juice concentrate 6 wt %, fructose 6 wt %, 0.4 wt % citrus pectin,citric acid 0.2 wt %, apple liquid spices 0.24 wt %, water 77.16 wt %.In other embodiments, the intake of red quinoa extract can be up to 10g/day. The red quinoa extract herein is produced with the methoddetailed in EXAMPLE 1. It should be noted that the concentrated applejuice 6 wt %, fructose 6 wt %, citrus pectin 0.4 wt %, citric acid 0.2wt %, apple liquid spices 0.24 wt % are meant to flavor and to enhancethe shelf life of the beverage.

Number of subjects: 30 subjects, 30-55 years of age.

Experimental Procedure

-   -   1. Collect 30 subjects. Before dunking the beverages, the        subjects' skin conditions (face cleaned, week 0) using the        corresponding instruments. Blood samples of the subjects were        also collected (under fasting conditions) to measure the content        of antioxidant enzymes in the body.    -   2. Next, randomly select 15 subjects as the placebo group and        the remaining 15 as the experimental group. The experimental        group bikes a bottle of the beverage daily (containing 5 g of        red quinoa extract) for a total of 56 days (i.e., 8 weeks). The        placebo group takes placebo beverages (same content as the        beverage drunk by the experimental group only that the 5 g red        quinoa extract was substituted with an equal amount of water).    -   3. The skin conditions of the subjects in the placebo group and        the experimental groups were measured at week 0, week 4, and        week 8. Their blood samples were also collected at week 0, week        4, and week 8. It should be noted that the temperature,        humidity, and other environmental factors of where the        measurements took place were the same at week 0, week 4, week 8        so as to reduce the impact of other factors on the skin        condition measurements. Moreover, during the clinical trial, the        subjects' diets and lifestyles were unchanged so as to reduce        other factors that may impact the content of in vivo        antioxidants.

Measurements

1. Skin Hydration, 2. Skin color, 3. Skin texture, 4 Crow's feet, 5.Antioxidant Enzyme Content

1. Skin Hydration

Purchased from the German company Courage+Khazaka electronic, the skinhydration probe Corneometer® CM825 (C+K MμLti Probe Adapter System,Germany) is used to detect facial skin hydration in both groups. Thevalues of the detection probe are based on capacitive measurement. Sincethe capacitance value of the skin changes with the skin's hydration, thedetection probe can analyze skin hydration by measuring skin capacitancevalues.

2. Skin Color

Purchased from the Irish company Miravex, the 3D analytical lens ofAntera 3D is used to detect facial skin color in both groups. Theinstrument first emits lights using its LED (light-emitting diodes) atdifferent wavelengths and angles and then collects the reflected lightsthat bounce off of the skin to analyze the skin color. Antera usescolorimeter to analyze color, which depends on the parameters L*a*b* inwhich L* refers to brightness: the a* axis represents the green-redcomponent, with green in the negative direction and red in the positivedirection; the b* axis represents the blue-yellow component, with bluein the negative direction and yellow in the positive direction.

For the difference (ΔE*_(ab)) color 1 (L*₁, a*₁, b*₁) and color 2 (L*₂,a*₂, b*₂) can be calculated by the following formula:

ΔE* _(ab)=√{square root over ((L* ₂ −L* ₁)²+(a* ₂ −a* ₁)²+(b* ₂ −b*₁)²)}

Since this example mainly concerns the whitening of the skin, only theL* value is observed and recorded. The higher the L* value is, thebrighter and hence the whiter the skin is.

3. Skin Texture

Skin texture is measured using the VISIA Digital Skin Detector(Canfield, US). The instrument uses visible light to capturehigh-resolution images of the skin and uses its built-in software toanalyze skin roughness. The higher the measured value is, the rougherthe skin is.

4. Crow's Feet Measurement

The Soft Plus Skin Detector (Callegari 1930) is used to measure the endof the subjects' eyes to evaluate crow's feet. The instrument detectsthe area of shades against standard light to quantify the depth ofcrow's feet.

5. Antioxidant Enzymes Measurement

First, the collected blood samples were transferred into a centrifugetube and centrifuged at 700-1000×g, 4° C. for 10 minutes. Aftercentrifugation, the upper plasma layer is removed, and 2 mL of the bloodcell layer (i.e., buffy coat) is placed in another centrifuge tube where2 mL of PBS is added and mixed to provide a diluted buffy coat.

Add 3 mL of the diluted buffy coat into another test tube and slowly addFicoll-Plague Plus solution (Sigma), and then centrifuged at arotational speed of 400 g for 40 minutes. After centrifugation, removethe supernatant liquid and transfer 2 mL-3 mL of the intermediate layer(i.e., the mononuclear cell layer) to a new centrifuge tube.

After washing the mononuclear cells with PBS 3 to 5 times, centrifuge itfor 10 minutes with 300 g. After centrifugation, the supernatant is thesample used for subsequent experiments.

Reference to the instructions provided by the Cayman Catalase Assay KitNo. 707002 (https://www.caymanchem.com/pdfs/707002.pdf). A summarizedprocedure is presented as follows:

Mix 10 μL of the standardized hydrogen peroxide formaldehyde enzyme(Cayman, No. 707014) with a buffer solution of 9.99 mL to obtain aformaldehyde solution of 4.25 mM. Take 7 tubes and label them from Athrough G and formulate them according to Table 3 to give the standardsolutions:

TABLE 3 Test Formaldehyde Buffer Solution Final Formaldehyde Tube (μL)(μL) Concentration (μM) A 0 1000 0 B 10 990 5 C 30 970 15 D 60 940 30 E90 910 45 F 120 880 60 G 150 850 75

This experiment is divided into three groups: the standard formaldehydegroup, the positive control group, and the experimental group. Thestandard formaldehyde group takes up seven wells, wherein each wellcontains 100 μL of buffer solution, 30 L of methanol, and 20 μL of thestandard solution (tube A to G); the positive control group takes up twowells, wherein each well contains 100 μL of buffer solution, 30 L ofmethanol, and 20 μL of the diluted antioxidant enzymes; while theexperimental group wells contain 100 μL of buffer solution, 30 μL ofmethanol and 20 μL of the experimental samples (i.e., the processedblood samples).

Add 20 μL of hydrogen peroxide to each well to start the reaction, andthen the wells were shaken at room temperature for 10 minutes.

Next, 30 μL of potassium hydroxide is added to terminate the reaction,and a 30 μL of dye (Catalase Purpalf, Cayman No. 707017) is added toeach well. The wells were then shaken at room temperature for 10minutes.

Then, add 10 μL Catalase Potassium Periodate (Cayman No. 707017) andshake wells at room temperature after 5 minutes.

Finally, read the absorbance values at 540 nm wavelength.

Catalase Content is calculated as follows:

First, subtract the absorbance values of each sample with the absorbancevalue of standard solution A and then plot the subtracted values. They-axis is the absorbance value (540 nm), while the x-axis is theconcentration of formaldehyde (μM). The calculation of the formaldehydeconcentration of the experimental group of:

Formaldehyde concentration (μM)=((absorbance value of experimentalgroup−y-intercept of the drawing made)/slope of the drawing made)*(0.17mL/0.02 mL)

Then calculate the enzyme activity of catalase:

Catalase activity=(sample formaldehyde concentration (μM))/(20min)×dilution ratio experiment=nmol/min/mL

The catalase content is set at 100% at week 0 for both the placebo andcontrol group to better present the relative catalase content of thegroups.

FIG. 7. shows the average skin hydration percentage at week 0, week 4,and week 8 of the placebo group and the experiment group. At week 4, 93%of the subjects had an increase in skin hydration, whereas, at week 8,100% of the subjects had an increase in skin hydration. When the testsubjects' average skin hydration percentage before beverage consumptionis 100%, the average skin hydration percentage after 4 weeks ofconsumption is 109.3%, while the average skin hydration percentage after8 weeks of consumption is 113.9%. In other words, the subjects in theexperimental group had an average increase of 9.3% and 13.9% in skinhydration, respectively, both reaching statistical significance. Theaverage skin hydration percentages of the experiment group in week 4 andweek 8 were also higher than those of the placebo group in week 4 andweek 8 by 5.1% and 6.6%, respectively. Thus, the present disclosed redquinoa extract can be used to moisturize the skin and maintain skinmoisture retention.

FIG. 8. shows the average skin color percentage at week 0, week 4, andweek 8 of the placebo group and the experiment group. At week 4, 93% ofthe subjects had an increase in skin color, whereas, at week 8, 100% ofthe subjects had an increase in skin color. When the test subjects'average skin color percentages before beverage consumption are 100%, theaverage skin color percentage after 4 weeks of consumption is 103.0%,while the average skin color percentage after 8 weeks of consumption is103.8%. In other words, the subjects in the experimental group had anaverage increase of 3.0% and 3.8% in skin color, respectively, bothreaching statistical significance. The average skin color percentages ofthe experiment group in week 4 and week 8 were also higher than those ofthe placebo group in week 4 and week 8 by 3.6% and 4.1%, respectively.Thus, the present disclosed red quinoa extract can be used to brightenand whiten skin color.

FIG. 9. shows the average skin texture percentage at week 0, week 4, andweek 8 of the placebo group and the experiment group. At week 4, 73% ofthe subjects had an increase in skin texture, whereas, at week 8, 83% ofthe subjects had an increase in skin texture. When the test subjects'average skin texture percentage before beverage consumption is 100%, theaverage skin texture percentage after 4 weeks of consumption is 91.1%,while the average skin texture percentage after 8 weeks of consumptionis 90.2%. In other words, the subjects in the experimental group had anaverage decrease of 8.9% and 9.8% in skin texture, respectively. Theaverage skin texture percentages of the experiment group in week 4 andweek 8 were also lower than those of the placebo group in week 4 andweek 8 by 10.6% and 18.1%, respectively. Thus, the present disclosed redquinoa extract can be used to improve skin texture and reduce theroughness of the skin

FIG. 10. shows the average crow's feet percentage at week 0, week 4, andweek 8 of the placebo group and the experiment group. At both week 4 andweek 8, 100% of the subjects had a decrease in crow's feet percentage.When the test subjects' average crow's feet percentage before beverageconsumption is 100%, the average crow's feet percentage after 4 weeks ofconsumption is 81.5%, while the average crow's feet percentage after 8weeks of consumption is 78.5%. In other words, the subjects in theexperimental group had an average decrease of 18.5% and 21.5% in crow'sfeet, respectively, both reaching statistical significance. The averagecrow's feet percentages of the experiment group in week 4 and week 8were also lower than those of the placebo group in week 4 and week 8 by9.9% and 8.9%, respectively. Thus, the present disclosed red quinoaextract can be used to soothe wrinkles

FIG. 11. shows the average in vivo antioxidant enzyme (catalase)percentage at week 0, week 4, and week 8 of the placebo group and theexperiment group. At week 8, 100% of the subjects had an increasedcatalase percentage. When the test subjects' average catalase percentagebefore beverage consumption is 100%, the average catalase percentageafter 8 weeks of consumption is 302.1%. In other words, the subjects inthe experimental group had an average increase of 202.1% in catalasepercentage, reaching statistical significance. The average catalasepercentages of the experiment group in week 8 were also higher thanthose of the placebo group in week 8 by 183.7%. Thus, the presentdisclosed red quinoa extract can lie used to enhance the content ofantioxidant enzymes, thereby achieving antioxidative properties in vivo.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, the disclosureis not for limiting the scope of the invention. Persons having ordinaryskill in the art may make various modifications and changes withoutdeparting from the scope and spirit of the invention. Therefore, thescope of the appended claims should not be limited to the description ofthe preferred embodiments described above.

What is claimed is:
 1. A method for skin whitening, comprisingcontacting a cell with a composition containing an effective amount of aChenopodium formosanum (Djulis) extract obtained by extraction of aChenopodium formosanum (Djulis) with an aqueous solvent.
 2. The methodof claim 1, wherein the Chenopodium formosanum (Djulis) extract reducesor inhibits melanin production in the cell.
 3. The method of claim 1,wherein the total flavonoid content of the Chenopodium formosanum(Djulis) extract equals or exceeds 1000 ppm.
 4. The method of claim 1,wherein the total polyphenol content of the Chenopodium formosanum(Djulis) extract equals or exceeds 400 ppm.
 5. The method of claim 1,wherein the concentration of the Chenopodium formosanum (Djulis) extractequals or exceeds 0.5 mg/mL.
 6. The method of claim 1, wherein thecomposition is a pharmaceutical composition, a health food composition,a food composition, or a cosmetic composition.
 7. A method formoisturizing skin, comprising contacting a cell with a compositioncontaining an effective amount of a Chenopodium formosanum (Djulis)extract obtained by extraction of a Chenopodium formosanum (Djulis) withan aqueous solvent.
 8. The method of claim 7, wherein the Chenopodiumformosanum (Djulis) extract shortens the gaps between a skin cell andanother skin cell.
 9. The method of claim 7, wherein the Chenopodiumformosanum (Djulis) extract promotes the expression of moisture-relatedgenes.
 10. The method of claim 7, wherein the total flavonoid content ofthe Chenopodium formosanum (Djulis) extract equals or exceeds 1000 ppm.11. The method of claim 7, wherein the total polyphenol content of theChenopodium formosanum (Djulis) extract equals or exceeds 400 ppm. 12.The method of claim 7, wherein the concentration of the Chenopodiumformosanum (Djulis) extract equals or exceeds 0.5 mg/mL.
 13. The methodof claim 7, wherein the composition is a pharmaceutical composition, ahealth food composition, a food composition, or a cosmetic composition.14. A method for scavenging free radicals in a cell, comprisingcontacting a cell with a composition containing an effective amount of aChenopodium formosanum (Djulis) extract obtained by extraction of aChenopodium formosanum (Djulis) with an aqueous solvent.
 15. The methodof claim 14, wherein the total flavonoid content of the Chenopodiumformosanum (Djulis) extract equals or exceeds 1000 ppm.
 16. The methodof claim
 14. wherein the total polyphenol content of the Chenopodiumformosanum (Djulis) extract equals or exceeds 400 ppm.
 17. The method ofclaim 14, wherein the concentration of the Chenopodium formosanum(Djulis) extract equals or exceeds 0.5 mg/mL.
 18. The method of claim14, wherein the composition is a pharmaceutical composition, a healthfood composition, a food composition, or a cosmetic composition.